Insect development and behavior are orchestrated by hormonal signaling cascades. We have identified a novel system of epitracheal glands, and associated peptidergic Inka cell, which secretes ecdysis-triggering hormones (ETHs). We will investigate the molecular physiology of this regulatory endocrine system in a genetic model (fruit fly, Drosophila) and a physiological model (moth, Manduca sexta). We previously demonstrated functions for ETHs in regulation of respiratory dynamics and a behavioral sequence associated with ecdysis, and established that ETH is an obligatory chemical signal for survival. The overall objective of this proposal is to provide a better understanding of how gene expression and hormones regulate developmental and behavioral processes. Long-range goals are to provide basic knowledge of chemical signaling processes in organisms applicable to development of therapeutics and management of insect vectors of human diseases. The first objective of the proposal is to define developmental, stage-specific functions for ETHs and to identify neuropeptides controlling their secretion. We will construct conditional promoters of ETH expression to rescue null mutant flies through each stage of development, in order to evaluate the physiological consequences of ETH deficits in later stages of ontogeny. Additional chemical signaling mechanisms, including the neuropeptide corazonin, will be evaluated for their regulation of ecdysis. For the second specific aim, we will examine the steroid regulation of cell-specific expression of ETHs and their release from Inka cells. Finally, our third specific aim will be to identify the nervous system receptor(s) for ETHs and the downstream neuronal targets of ETH action in the CNS. The ecdysis sequence is an excellent model for relating steroid and peptide signaling processes to physiology and behavior. Drosophila offers powerful genetic tools for deletion and conditional expression of key genes involved in these processes, while the moth Manduca offers advantages for endocrinological and physiological manipulations. Use of these models will allow us to pose testable hypotheses at many levels, and to mobilize genetic and cellular approaches appropriate to each objective. The simplicity of the epitracheal endocrine system, composed of glands containing only 1-4 cells, provides an excellent model for examination of endocrine function at the cellular, developmental and molecular levels. Since ecdysis is crucial for successful development of insects and other arthropods, understanding the regulatory mechanisms which underlie these precisely timed and synchronized processes should allow in the future for more sophisticated approaches to the study of basic physiology and behavior. [unreadable] [unreadable]